Abstract
SummaryBackgroundBilateral symmetry during vertebrate development is broken at the left-right organizer (LRO) by ciliary motility and the resultant directional flow of extracellular fluid. However, how ciliary motility is perceived and transduced into asymmetrical intracellular signaling at the LRO remains controversial. Previous work has indicated that sensory cilia and polycystin-2 (Pkd2), a cation channel, are required for sensing ciliary motility, yet their function and the molecular mechanism linking both to left-right signaling cascades is unknown.ResultsHere, we report novel intraciliary calcium oscillations (ICOs) at the LRO that connect ciliary sensation of ciliary motility to downstream left-right signaling. Utilizing cilia-targeted genetically-encoded calcium indicators in live zebrafish embryos, we show that ICOs depend on Pkd2 and are left-biased at the LRO in response to ciliary motility. Asymmetric ICOs occur with onset of LRO ciliary motility, thus representing the earliest known LR asymmetric molecular signal. Suppression of ICOs using a cilia-targeted calcium sink demonstrates that they are essential for LR development.ConclusionsThese findings demonstrate that intraciliary calcium initiates LR development and identify cilia as a functional ion signaling compartment connecting ciliary motility and flow to molecular LR signaling.
Highlights
In vertebrates, cilia and polycystins are essential for the development of left-right (LR) asymmetry
One of the earliest observed molecular LR asymmetries is an increase in cytoplasmic mesendodermal calcium at the left side of the left-right organizer (LRO) that depends on both ciliary motility and the cation channel polycystin-2 (PKD2) and has been observed in mouse and zebrafish [9,10,11, 15]
Similar to inhibition of intraciliary calcium by arl13b-Pvalb, morpholino knockdown of pkd2 resulted in a loss of left-sided mesendodermal calcium (Figures 4E–4G; Movie S6). These results demonstrate that intraciliary calcium is upstream of asymmetric mesendodermal calcium at the LRO and that Pkd2 modulates intraciliary calcium to affect LR mesendodermal calcium asymmetry
Summary
Cilia and polycystins are essential for the development of left-right (LR) asymmetry. One of the earliest observed molecular LR asymmetries is an increase in cytoplasmic mesendodermal calcium at the left side of the LRO that depends on both ciliary motility and the cation channel polycystin-2 (PKD2) and has been observed in mouse and zebrafish [9,10,11, 15]. In cultured renal epithelial cells, mechanical stress on the cilium, either by direct pipette manipulation or application of laminar fluid flow, results in a rise in cytosolic calcium that depends on both PKD2 and its binding partner PKD1 [19, 21,22,23], suggesting that the polycystin complex can function as a mechanosensitive calcium channel. The mechanism linking ciliary motility, sensation, polycystins, calcium, and the development of molecular and morphological LR asymmetry remains a mystery
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